IRF8-driven reprogramming of the immune microenvironment enhances anti-tumor adaptive immunity and reduces immunosuppression in murine glioblastoma.

Background: Glioblastoma (GBM) has a highly immunosuppressive tumor immune microenvironment (TIME), largely mediated by myeloid-derived suppressor cells (MDSCs). Here, we utilized a retroviral replicating vector (RRV) to deliver Interferon Regulatory Factor 8 (IRF8), a master regulator of type 1 conventional dendritic cell (cDC1) development, in a syngeneic murine GBM model. We hypothesized that RRV-mediated delivery of IRF8 could "reprogram" intratumoral MDSCs into antigen-presenting cells (APCs) and thereby restore T-cell responses. Methods: Effects of RRV-IRF8 on survival and tumor growth kinetics were examined in the SB28 murine GBM model. Immunophenotype was analyzed by flow cytometry and gene expression assays. We assayed functional immunosuppression and antigen presentation by ex vivo T-cell-myeloid co-culture. Results: Mice with RRV-IRF8 pre-transduced intracerebral tumors had significantly longer survival and slower tumor growth compared to controls. RRV-IRF8 treated tumors exhibited significant enrichment of cDC1s and CD8+ T-cells. Additionally, myeloid cells derived from RRV-IRF8 tumors showed decreased expression of the immunosuppressive markers Arg1 and IDO1 and demonstrated reduced suppression of naïve T-cell proliferation in ex vivo co-culture, compared to controls. Furthermore, DCs from RRV-IRF8 tumors showed increased antigen presentation compared to those from control tumors. In vivo treatment with azidothymidine (AZT), a viral replication inhibitor, showed that IRF8 transduction in both tumor and non-tumor cells is necessary for survival benefit, associated with a reprogrammed, cDC1- and CD8 T-cell-enriched TIME. Conclusions: Our results indicate that reprogramming of glioma-infiltrating myeloid cells by in vivo expression of IRF8 may reduce immunosuppression and enhance antigen presentation, achieving improved tumor control.

Challenges in the discovery of tumor-specific alternative splicing-derived cell-surface antigens in glioma.

Despite advancements in cancer immunotherapy, solid tumors remain formidable challenges. In glioma, profound inter- and intra-tumoral heterogeneity of antigen landscape hampers therapeutic development. Therefore, it is critical to consider alternative sources to expand the repertoire of targetable (neo-)antigens and improve therapeutic outcomes. Accumulating evidence suggests that tumor-specific alternative splicing (AS) could be an untapped reservoir of antigens. In this study, we investigated tumor-specific AS events in glioma, focusing on those predicted to generate major histocompatibility complex (MHC)-presentation-independent, cell-surface antigens that could be targeted by antibodies and chimeric antigen receptor-T cells. We systematically analyzed bulk RNA-sequencing datasets comparing 429 tumor samples (from The Cancer Genome Atlas) and 9166 normal tissue samples (from the Genotype-Tissue Expression project), and identified 13 AS events in 7 genes predicted to be expressed in more than 10% of the patients, including PTPRZ1 and BCAN, which were corroborated by an external RNA-sequencing dataset. Subsequently, we validated our predictions and elucidated the complexity of the isoforms using full-length transcript amplicon sequencing on patient-derived glioblastoma cells. However, analyses of the RNA-sequencing datasets of spatially mapped and longitudinally collected clinical tumor samples unveiled remarkable spatiotemporal heterogeneity of the candidate AS events. Furthermore, proteomics analysis did not reveal any peptide spectra matching the putative antigens. Our investigation illustrated the diverse characteristics of the tumor-specific AS events and the challenges of antigen exploration due to their notable spatiotemporal heterogeneity and elusive nature at the protein levels. Redirecting future efforts toward intracellular, MHC-presented antigens could offer a more viable avenue.

Enhancing CAR-T cell metabolism to overcome hypoxic conditions in the brain tumor microenvironment.

The efficacy of chimeric antigen receptor T cell (CAR-T) therapy has been limited against brain tumors to date. CAR-T cells infiltrating syngeneic intracerebral SB28 EGFRvIII gliomas revealed impaired mitochondrial ATP production and a markedly hypoxic status compared with ones migrating to subcutaneous tumors. Drug screenings to improve metabolic states of T cells under hypoxic conditions led us to evaluate the combination of the AMPK activator metformin and the mTOR inhibitor rapamycin (Met+Rap). Met+Rap-pretreated mouse CAR-T cells showed activated PPAR-γ coactivator 1α (PGC-1α) through mTOR inhibition and AMPK activation, and a higher level of mitochondrial spare respiratory capacity than those pretreated with individual drugs or without pretreatment. Moreover, Met+Rap-pretreated CAR-T cells demonstrated persistent and effective antiglioma cytotoxic activities in the hypoxic condition. Furthermore, a single intravenous infusion of Met+Rap-pretreated CAR-T cells significantly extended the survival of mice bearing intracerebral SB28 EGFRvIII gliomas. Mass cytometric analyses highlighted increased glioma-infiltrating CAR-T cells in the Met+Rap group, with fewer Ly6c+CD11b+ monocytic myeloid-derived suppressor cells in the tumors. Finally, human CAR-T cells pretreated with Met+Rap recapitulated the observations with murine CAR-T cells, demonstrating improved functions under in vitro hypoxic conditions. These findings advocate for translational and clinical exploration of Met+Rap-pretreated CAR-T cells in human trials.

Challenges in the discovery of tumor-specific alternative splicing-derived cell-surface antigens in glioma.

Background: Despite advancements in cancer immunotherapy, solid tumors remain formidable challenges. In glioma, profound inter-and intra-tumoral heterogeneity of antigen landscape hampers therapeutic development. Therefore, it is critical to consider alternative sources to expand the repertoire of targetable (neo-)antigens and improve therapeutic outcomes. Accumulating evidence suggests that tumor-specific alternative splicing (AS) could be an untapped reservoir of neoantigens. Results: In this study, we investigated tumor-specific AS events in glioma, focusing on those predicted to generate major histocompatibility complex (MHC)-presentation-independent, cell-surface neoantigens that could be targeted by antibodies and chimeric antigen receptor (CAR)-T cells. We systematically analyzed bulk RNA-sequencing datasets comparing 429 tumor samples (from The Cancer Genome Atlas [TCGA]) and 9,166 normal tissue samples (from the Genotype-Tissue Expression project [GTEx]), and identified 13 AS events in 7 genes predicted to be expressed in more than 10% of the patients, including PTPRZ1 and BCAN , which were corroborated by an external RNA-sequencing dataset. Subsequently, we validated our predictions and elucidated the complexity of the isoforms using full-length transcript amplicon sequencing on patient-derived glioblastoma cells. However, analyses of the RNA-sequencing datasets of spatially mapped and longitudinally collected clinical tumor samples unveiled remarkable spatiotemporal heterogeneity of the candidate AS events. Furthermore, proteomics analysis did not reveal any peptide spectra matching the putative neoantigens. Conclusions: Our investigation illustrated the diverse characteristics of the tumor-specific AS events and the challenges of antigen exploration due to their notable spatiotemporal heterogeneity and elusive nature at the protein levels. Redirecting future efforts toward intracellular, MHC-presented antigens could offer a more viable avenue.

Enhancing CAR-T Cell Metabolism to Overcome Hypoxic Conditions in the Brain Tumor Microenvironment.

The efficacy of chimeric antigen receptor (CAR)-T therapy has been limited against brain tumors to date. CAR-T cells infiltrating syngeneic intracerebral SB28-EGFRvIII glioma revealed impaired mitochondrial ATP production and a markedly hypoxic status compared to ones migrating to subcutaneous tumors. Drug screenings to improve metabolic states of T cells under hypoxic conditions led us to evaluate the combination of AMPK activator Metformin and the mTOR inhibitor Rapamycin (Met+Rap). Met+Rap-pretreated mouse CAR-T cells showed activated PPAR-gamma coactivator 1α (PGC-1α) through mTOR inhibition and AMPK activation, and a higher level of mitochondrial spare respiratory capacity than those pretreated with individual drugs or without pretreatment. Moreover, Met+Rap-pretreated CAR-T cells demonstrated persistent and effective anti-glioma cytotoxic activities in the hypoxic condition. Furthermore, a single intravenous infusion of Met+Rap-pretreated CAR-T cells significantly extended the survival of mice bearing intracerebral SB28-EGFRvIII gliomas. Mass cytometric analyses highlighted increased glioma-infiltrating CAR-T cells in the Met+Rap group with fewer Ly6c+ CD11b+ monocytic myeloid-derived suppressor cells in the tumors. Finally, human CAR-T cells pretreated with Met+Rap recapitulated the observations with murine CAR-T cells, demonstrating improved functions in vitro hypoxic conditions. These findings advocate for translational and clinical exploration of Met+Rap-pretreated CAR-T cells in human trials.

Tumor-wide RNA splicing aberrations generate immunogenic public neoantigens.

T-cell-mediated immunotherapies are limited by the extent to which cancer-specific antigens are homogenously expressed throughout a tumor. We reasoned that recurrent splicing aberrations in cancer represent a potential source of tumor-wide and public neoantigens, and to test this possibility, we developed a novel pipeline for identifying neojunctions expressed uniformly within a tumor across diverse cancer types. Our analyses revealed multiple neojunctions that recur across patients and either exhibited intratumor heterogeneity or, in some cases, were tumor-wide. We identified CD8+ T-cell clones specific for neoantigens derived from tumor-wide and conserved neojunctions in GNAS and RPL22 , respectively. TCR-engineered CD8 + T-cells targeting these mutations conferred neoantigen-specific tumor cell eradication. Furthermore, we revealed that cancer-specific dysregulation in splicing factor expression leads to recurrent neojunction expression. Together, these data reveal that a subset of neojunctions are both intratumorally conserved and public, providing the molecular basis for novel T-cell-based immunotherapies that address intratumoral heterogeneity.

Glioma-neuronal circuit remodeling induces regional immunosuppression.

Neuronal activity-driven mechanisms impact glioblastoma cell proliferation and invasion 1-7 , and glioblastoma remodels neuronal circuits 8,9 . Distinct intratumoral regions maintain functional connectivity via a subpopulation of malignant cells that mediate tumor-intrinsic neuronal connectivity and synaptogenesis through their transcriptional programs 8 . However, the effects of tumor-intrinsic neuronal activity on other cells, such as immune cells, remain unknown. Here we show that regions within glioblastomas with elevated connectivity are characterized by regional immunosuppression. This was accompanied by different cell compositions and inflammatory status of tumor-associated macrophages (TAMs) in the tumor microenvironment. In preclinical intracerebral syngeneic glioblastoma models, CRISPR/Cas9 gene knockout of Thrombospondin-1 (TSP-1/ Thbs1 ), a synaptogenic factor critical for glioma-induced neuronal circuit remodeling, in glioblastoma cells suppressed synaptogenesis and glutamatergic neuronal hyperexcitability, while simultaneously restoring antigen-presentation and pro-inflammatory responses. Moreover, TSP-1 knockout prolonged survival of immunocompetent mice harboring intracerebral syngeneic glioblastoma, but not of immunocompromised mice, and promoted infiltrations of pro-inflammatory TAMs and CD8+ T-cells in the tumor microenvironment. Notably, pharmacological inhibition of glutamatergic excitatory signals redirected tumor-associated macrophages toward a less immunosuppressive phenotype, resulting in prolonged survival. Altogether, our results demonstrate previously unrecognized immunosuppression mechanisms resulting from glioma-neuronal circuit remodeling and suggest future strategies targeting glioma-neuron-immune crosstalk may open up new avenues for immunotherapy.

Immunotherapy Approaches in Isocitrate-Dehydrogenase-Mutant Low-Grade Glioma.

Low-grade gliomas (LGGs) are slow-growing tumors in the central nervous system (CNS). Patients characteristically show the onset of seizures or neurological deficits due to the predominant LGG location in high-functional brain areas. As a molecular hallmark, LGGs display mutations in the isocitrate dehydrogenase (IDH) enzymes, resulting in an altered cellular energy metabolism and the production of the oncometabolite D-2-hydroxyglutarate. Despite the remarkable progress in improving the extent of resection and adjuvant radiotherapy and chemotherapy, LGG remains incurable, and secondary malignant transformation is often observed. Therefore, novel therapeutic approaches are urgently needed. In recent years, immunotherapeutic strategies have led to tremendous success in various cancer types, but the effect of immunotherapy against glioma has been limited due to several challenges, such as tumor heterogeneity and the immunologically "cold" tumor microenvironment. Nevertheless, recent preclinical and clinical findings from immunotherapy trials are encouraging and offer a glimmer of hope for treating IDH-mutant LGG patients. Here, we aim to review the lessons learned from trials involving vaccines, T-cell therapies, and IDH-mutant inhibitors and discuss future approaches to enhance the efficacy of immunotherapies in IDH-mutant LGG.

Correction: Gallus et al. Immunotherapy Approaches in Isocitrate-Dehydrogenase-Mutant Low-Grade Glioma. Cancers 2023, 15, 3726.

It has come to our attention that the previously published manuscript contained an outdated iteration of Table 1 [...].

Efficacy of cancer-specific anti-podoplanin CAR-T cells and oncolytic herpes virus G47Δ combination therapy against glioblastoma.

Glioblastoma is a devastating malignant brain tumor with a poor prognosis despite standard therapy. Podoplanin (PDPN), a type I transmembrane mucin-like glycoprotein that is overexpressed in various cancers, is a potential therapeutic target for the treatment of glioblastoma. We previously reported the efficacy of chimeric antigen receptor (CAR)-T cells using an anti-pan-PDPN monoclonal antibody (mAb; NZ-1)-based third-generation CAR in a xenograft mouse model. However, NZ-1 also reacted with PDPN-expressing normal cells, such as lymphatic endothelial cells, pulmonary alveolar type I cells, and podocytes. To overcome possible on-target-off-tumor effects, we produced a cancer-specific mAb (CasMab, LpMab-2)-based CAR. LpMab-2 (Lp2) reacted with PDPN-expressing cancer cells but not with normal cells. In this study, Lp2-CAR-transduced T cells (Lp2-CAR-T) specifically targeted PDPN-expressing glioma cells while sparing the PDPN-expressing normal cells. Lp2-CAR-T also killed patient-derived glioma stem cells, demonstrating its clinical potential against glioblastoma. Systemic injection of Lp2-CAR-T cells inhibited the growth of a subcutaneous glioma xenograft model in immunodeficient mice. Combination therapy with Lp2-CAR-T and oncolytic virus G47Δ, a third-generation recombinant herpes simplex virus (HSV)-1, further inhibited the tumor growth and improved survival. These findings indicate that the combination therapy of Lp2-CAR-T cells and G47Δ may be a promising approach to treat glioblastoma.

Inhibition of D-2HG leads to upregulation of a proinflammatory gene signature in a novel HLA-A2/HLA-DR1 transgenic mouse model of IDH1R132H-expressing glioma.

BACKGROUND: Long-term prognosis of WHO grade II, isocitrate dehydrogenase (IDH)-mutated low-grade glioma (LGG) is poor due to high risks of recurrence and malignant transformation into high-grade glioma. Immunotherapy strategies are attractive given the relatively intact immune system of patients with LGG and the slow tumor growth rate. However, accumulation of the oncometabolite D-2-hydroxyglutarate (D-2HG) in IDH-mutated gliomas leads to suppression of inflammatory pathways in the tumor microenvironment, thereby contributing to the 'cold' tumor phenotype. Inhibiting D-2HG production presents an opportunity to generate a robust antitumor response following tumor antigen vaccination and immune checkpoint blockade. METHODS: An IDH1R132H glioma model was created in syngeneic HLA-A2/HLA-DR1-transgenic mice, allowing us to evaluate the vaccination with the human leukocyte antigens (HLA)-DR1-restricted, IDH1R132H mutation-derived neoepitope. The effects of an orally available inhibitor of mutant IDH1 and IDH2, AG-881, were evaluated as monotherapy and in combination with the IDH1R132H peptide vaccination or anti-PD-1 immune checkpoint blockade. RESULTS: The HLA-A2/HLA-DR1-syngeneic IDH1R132H cell line expressed the IDH1 mutant protein and formed D-2HG producing orthotopic gliomas in vivo. Treatment of tumor-bearing mice with AG-881 resulted in a reduction of D-2HG levels in IDH1R132H glioma cells (10 fold) and tumor-associated myeloid cells, which demonstrated high levels of intracellular D-2HG in the IDH1R132H gliomas. AG-881 monotherapy suppressed the progression of IDH1R132H gliomas in a CD4+ and CD8+ cell-dependent manner, enhanced proinflammatory IFNγ-related gene expression, and increased the number of CD4+ tumor-infiltrating T-cells. Prophylactic vaccination with the HLA-DR1-restricted IDH1R132H peptide or tumor-associated HLA-A2-restricted peptides did not enhance survival of tumor-bearing animals; however, vaccination with both HLA-A2-IDH1R132H and DR1-IDH1R132H peptides in combination with the IDH inhibitor significantly prolonged survival. Finally, tumor-bearing mice treated with both AG-881 and a PD-1 blocking antibody demonstrated improved survival when compared with either treatment alone. CONCLUSION: The development of effective IDH1R132H-targeting vaccine may be enhanced by integration with HLA class I-restricted cytotoxic T cell epitopes and AG-881. Our HLA-A2/HLA-DR1-syngeneic IDH1R132H glioma model should allow us to evaluate key translational questions related to the development of novel strategies for patients with IDH-mutant glioma.

Novel EGFRvIII-CAR transgenic mice for rigorous preclinical studies in syngeneic mice.

BACKGROUND: Rigorous preclinical studies of chimeric antigen receptor (CAR) immunotherapy will require large quantities of consistent and high-quality CAR-transduced T (CART) cells that can be used in syngeneic mouse glioblastoma (GBM) models. To this end, we developed a novel transgenic (Tg) mouse strain with a fully murinized CAR targeting epidermal growth factor receptor variant III (EGFRvIII). METHODS: We first established the murinized version of EGFRvIII-CAR and validated its function using a retroviral vector (RV) in C57BL/6J mice bearing syngeneic SB28 GBM expressing EGFRvIII. Next, we created C57BL/6J-background Tg mice carrying the anti-EGFRvIII-CAR downstream of a Lox-Stop-Lox cassette in the Rosa26 locus. We bred these mice with CD4-Cre Tg mice to allow CAR expression on T cells and evaluated the function of the CART cells both in vitro and in vivo. To inhibit immunosuppressive myeloid cells within SB28 GBM, we also evaluated a combination approach of CART and an anti-EP4 compound (ONO-AE3-208). RESULTS: Both RV- and Tg-CART cells demonstrated specific cytotoxic activities against SB28-EGFRvIII cells. A single intravenous infusion of EGFRvIII-CART cells prolonged the survival of glioma-bearing mice when preceded by a lymphodepletion regimen with recurrent tumors displaying profound EGFRvIII loss. The addition of ONO-AE3-208 resulted in long-term survival in a fraction of CART-treated mice and those survivors demonstrated delayed growth of subcutaneously re-challenged both EGFRvIII+ and parental EGFRvIII- SB28. CONCLUSION: Our new syngeneic CAR Tg mouse model can serve as a useful tool to address clinically relevant questions and develop future immunotherapeutic strategies.

Reliability of IDH1-R132H and ATRX and/or p53 immunohistochemistry for molecular subclassification of Grade 2/3 gliomas.

Since the World Health Organization 2016 classification (2016 WHO), genetic status has been incorporated into the diagnosis of Grade 2/3 gliomas (lower-grade gliomas). Therefore, immunohistochemistry (IHC) of IDH1-R132H, ATRX, and p53 have been used in place of genetic status. We report the associations between histological findings, IHC, and genetic status. We performed IHC of IDH1-R132H, ATRX, and p53 in 76 lower-grade gliomas and discussed its validity based on the 2016 WHO and the upcoming 2021 WHO classification. The sensitivity and specificity of anti-ATRX, p53, and IDH1-R132H IHC were 40.9%/98.1%, 78.6%/85.4%, and 90.5%/84.6%, respectively. Among 21 IDH1-mutant gliomas without 1p/19q codeletion, two gliomas (9.5%) mimicked the so-called classic for oligodendroglioma (CFO) in their morphology. Of the 42 gliomas with 1p/19q codeletion, four cases were difficult to diagnose as oligodendroglioma through morphological examination. Moreover, there were three confusing cases with ATRX mutations but with retained ATRX-IHC positivity. The lessons learned from this study are as follows: (1) ATRX-IHC and p53-IHC should be supplementary to morphological diagnosis, (2) rare IDH mutations other than IDH1 R132H should be considered, and (3) there is no complete alternative test to detect molecular features of glioblastoma under the 2021 WHO classification.

Tumor antigens in glioma.

Immunotherapy applications to glioblastoma represent a new treatment frontier. Antigen-targeted immunotherapy approaches hold enormous potential to elicit antigen-specific anti-tumor effects in central nervous system tumors. Still, the paucity of effective antigen targets remains a significant obstacle in safely and effectively treating glioblastoma and other malignant gliomas with relatively low mutation loads. In this review, we highlight the current understanding of and development of immunotherapy to target 1) shared non-mutant antigens 2) shared mutant antigens (neoantigens) derived from cancer-specific mutations 3) personalized neoantigens derived from tumor-specific genetic alterations containing de novo peptide sequences and 4) virus-derived antigens. We also discuss strategies to enhance tumor immunogenicity and neoantigen prediction. Spatial heterogeneity remains a formidable challenge for immunotherapy of glioma; recent advances in targeting multiple antigens and refining the antigen selection pipeline hold great promise to turn the tide against glioma.

Pathogenic Epigenetic Consequences of Genetic Alterations in IDH-Wild-Type Diffuse Astrocytic Gliomas.

Gliomas are classified by combining histopathologic and molecular features, including isocitrate dehydrogenase (IDH) status. Although IDH-wild-type diffuse astrocytic glioma (DAG) shows a more aggressive phenotype than IDH-mutant type, lack of knowledge regarding relevant molecular drivers for this type of tumor has hindered the development of therapeutic agents. Here, we examined human IDH-wild-type DAGs and a glioma mouse model with a mosaic analysis with double markers (MADM) system, which concurrently lacks p53 and NF1 and spontaneously develops tumors highly comparable with human IDH-wild-type DAG without characteristic molecular features of glioblastoma (DAG-nonMF). During tumor formation, enhancer of zeste homolog (EZH2) and the other polycomb repressive complex 2 (PRC2) components were upregulated even at an early stage of tumorigenesis, together with an increased number of genes with H3K27me3 or H3K27me3 and H3K4me3 bivalent modifications. Among the epigenetically dysregulated genes, frizzled-8 (Fzd8), which is known to be a cancer- and stem cell reprogramming-related gene, was gradually silenced during tumorigenesis. Genetic and pharmacologic inhibition of EZH2 in MADM mice showed reactivation of aberrant H3K27me3 target genes, including Fzd8, together with significant reduction of tumor size. Our study clarifies a pathogenic molecular pathway of IDH-wild-type DAG-nonMF that depends on EZH2 activity and provides a strong rationale for targeting EZH2 as a promising therapeutic approach for this type of glioma. SIGNIFICANCE: EZH2 is involved in the generation of IDH-wild-type diffuse astrocytic gliomas and is a potential therapeutic target for this type of glioma. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/79/19/4814/F1.large.jpg.

Aberrant Active cis-Regulatory Elements Associated with Downregulation of RET Finger Protein Overcome Chemoresistance in Glioblastoma.

RET finger protein (RFP) forms a complex with histone deacetylase 1, resulting in aberrant deacetylation of H3K27ac and dysregulation of cis-regulatory elements. We evaluated the modulatory effects of RFP knockdown on cis-regulatory elements, gene expression, and chemosensitivity to temozolomide both in glioblastoma cells and in an intracranial glioblastoma model. The combination of RFP knockdown and temozolomide treatment markedly suppressed the glioblastoma cell growth due to oxidative stress and aberrant cell cycle and increased survival time in mice with glioblastoma. ChIP-seq and RNA-seq revealed that RFP knockdown increased or decreased activity of numerous cis-regulatory elements that lie adjacent to genes that control functions such as apoptosis, mitosis, DNA replication, and cell cycle: FOXO1, TBP2, and PARPBP. This study suggests that RFP contributes to chemoresistance via aberrant deacetylation of histone H3 at K27, whereas dysregulation of RFP-associated cis-regulatory elements in glioma and RFP knockdown combined with temozolomide is an effective treatment strategy for lethal glioma.

Immunohistochemical ATRX expression is not a surrogate for 1p19q codeletion.

The IDH-mutant and 1p/19q co-deletion (1p19q codel) provides significant diagnostic and prognostic value in lower-grade gliomas. As ATRX mutation and 1p19q codel are mutually exclusive, ATRX immunohistochemistry (IHC) may substitute for 1p19q codel, but this has not been comprehensively examined. In the current study, we performed ATRX-IHC in 78 gliomas whose ATRX statuses were comprehensively determined by whole exome sequencing. Among the 60 IHC-positive and 18 IHC-negative cases, 86.7 and 77.8% were ATRX-wildtype and ATRX-mutant, respectively. ATRX mutational patterns were not consistent with ATRX-IHC. If our cohort had only used IDH status and IHC-based ATRX expression for diagnosis, 78 tumors would have been subtyped as 48 oligodendroglial tumors, 16 IDH-mutant astrocytic tumors, and 14 IDH-wildtype astrocytic tumors. However, when the 1p19q codel test was performed following ATRX-IHC, 8 of 48 ATRX-IHC-positive tumors were classified as "1p19q non-codel" and 3 of 16 ATRX-IHC-negative tumors were classified as "1p19q codel"; a total of 11 tumors (14%) were incorrectly classified. In summary, we observed dissociation between ATRX-IHC and actual 1p19q codel in 11 of 64 IDH-mutant LGGs. In describing the complex IHC expression of ATRX somatic mutations, our results indicate the need for caution when using ATRX-IHC as a surrogate of 1p19q status.

A novel high-sensitivity assay to detect a small fraction of mutant IDH1 using droplet digital PCR.

Detection of mutations in the isocitrate dehydrogenase 1 (IDH1) gene is useful for accurate diagnosis of lower grade gliomas, as described in the 2016 World Health Organization classification of tumors of the central nervous system. Conventional analysis tools, including Sanger DNA sequencing and immunohistochemistry, might fail to detect a small fraction of mutant IDH1 owing to their limited sensitivity. Considering that lower grade gliomas are infiltrative in nature, a highly sensitive detection assay for IDH1 mutation is required for their accurate diagnosis. In this study, we successfully established a droplet digital PCR (ddPCR) system to detect a small fraction of IDH1 mutation. We could detect 0.05% of mutant IDH1 allele in 30 ng DNA. Using this assay, we could detect a small fraction of mutant IDH1 in a glioma case, identified as a wildtype tumor according to the conventional assays. Additionally, in a small amount of DNA derived from the cerebrospinal fluid, we could detect an IDH1 mutation. In conclusion, the ddPCR system is useful to identify a small fraction of IDH1 mutation in diffuse infiltrative gliomas. This might be useful for precision medicine of these gliomas in the near future and also for the non-invasive diagnosis of these gliomas.

Remote ischemic preconditioning protects human neural stem cells from oxidative stress.

In previous clinical trials, we showed that remote ischemic preconditioning (rIPC) reduced myocardial damage in children undergoing treatment for congenital heart defects and postoperative renal failure in patients undergoing abdominal aortic aneurysm surgery. In rabbit experiments, pre-treatment with plasma and plasma dialysate (obtained using 15-kDa cut-off dialysis membrane) from donor rabbits subjected to rIPC similarly protected against cardiac infarction. However, the protective substances containing in rIPC plasma have been unknown. In the present study, we showed that rIPC plasma exerted anti-apoptotic and anti-oxidative effects on human neural stem cells under oxygen glucose deprivation (OGD) that mimics brain ischemia. Additionally, we applied the sample to the liquid chromatography integrated with mass spectrometry to identify candidate key molecules in the rIPC plasma and determine its role in protecting neural stem cells from OGD-induced cell death. Thioredoxin increased significantly after rIPC compared to pre-IPC. Pretreatment with thioredoxin, the antioxidant protein, markedly protected human neural stem cells from OGD-induced cell death. The effect of thioredoxin on brain ischemia in animals should be further evaluated. However, the present study first evaluated the effect of rIPC in the ischemic cellular model.